Activation of the NO-cGMP pathway is associated with myocardial protection against ischemia. During ischemia, function of this pathway is disturbed. Little is known about the effects of supplements such as Red Palm Oil (RPO) on the myocardial NO- cGMP- signalling pathway. RPO consists of saturated (SFAs), monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids and is an antioxidant rich in natural B-carotene and vitamin E (tocopherols and tocotrienols). This study determined whether dietary RPO-supplemention protects against the consequences of ischemia and identified a possible mechanism for this protection. Long-Evans rats were fed a control diet or control diet plus 7g RPO per kg diet for six weeks. Hearts were excised and mounted on a working heart perfusion apparatus. Cardiac function was measured before and after hearts were subjected to 25 minutes of global ischemia. Left ventricular systolic (LVSP) and diastolic pressure (LVDP), coronary flow (CF), heart rate (HR) and aortic output (AO) were measured. To assess NO-cGMP pathway activity, hearts subjected to the same conditions, were freeze-clamped and analysed for tissue cAMP and cGMP levels using a RIA method. Furthermore, composition of myocardial phospholipid fatty acids by gaschromatography and blood samples were collected for serum lipid determinations. The percentage aortic output recovery of hearts supplemented with RPO was 72.9 ± 3.43 % vs 55.4 ± 2.48 % for controls (P &lt; 0.05). Ten minutes into ischemia the cGMP levels of the RPO-supplementation group were significantly higher than the control group (26.5 ± 2.78 pmol/g vs 10.1 ± 1.78 pmol/g. Total myocardial PUFA content in hearts supplemented with RPO increased from 54.45 ± 1.11% before ischemia to 59.03 ± 0.30 % after ischemia (P&lt;0.05). Results demonstrated that RPO-supplementation protected hearts against the consequences of ischemia/reperfusion injury. These findings suggest that dietary RPO protects via the NO-cGMP pathway and/or changes in PUFA composition during ischemia/reperfusion.